A rad50 germline mutation induces tumorigenesis and ataxia-telangiectasia phenotype in a transparent medaka model

The MRE11A-RAD50-NBS1 complex activates the ataxia-telangiectasia mutated (ATM) pathway and plays a central role in genome homeostasis. The association of RAD50 mutations with disease remains unclear; hence, we adopted a medaka rad50 mutant to demonstrate the significance of RAD50 mutation in pathogenesis using the medaka as an experimental animal. A 2-base pair deletion in the rad50 gene was introduced into transparent STIII medaka using the CRISPR/Cas9 system. The mutant was analyzed histologically for tumorigenicity and hindbrain quality, as well as for swimming behavior, to compare with existing ATM-, MRE11A-, and NBS1-mutation-related pathology. Our results revealed that the medaka rad50 mutation concurrently reproduced tumorigenesis (8 out of 10 rad50Δ2/+ medaka), had a decrease in median survival time (65.7 ± 1.1 weeks in control vs. 54.2 ± 2.6 weeks in rad50Δ2/+ medaka, p = 0.001, Welch’s t-test), exhibited semi-lethality in rad50Δ2/Δ2 medaka and most of the major ataxia-telangiectasia phenotypes, including ataxia (rheotaxis ability was lower in rad50Δ2/+ medaka than in the control, Mann–Whitney U test, p < 0.05), and telangiectasia (6 out of 10 rad50Δ2/+ medaka). The fish model may aid in further understanding the tumorigenesis and phenotype of ataxia-telangiectasia-related RAD50 germline mutations and in developing novel therapeutic strategies against RAD50 molecular disorders.


Introduction
RAD50 is a molecule that, along with meiotic recombination 11 (MRE11) and Nijmegen breakage syndrome 1 (NBS1), constitutes the MRE11-RAD50-NBS1 (MRN) complex [1]. The MRN complex is conserved in all eukaryotes and plays a central role in maintaining genome homeostasis, particularly by repairing DNA double-strand breaks (DSBs) [2,3] that occur during meiosis, homologous recombination, non-homologous end joining, DNA damage [4], and telomere management [5]. This complex activates ataxia-telangiectasia mutated (ATM) kinase [6], which initiates mechanisms associated with DNA repair, cell cycle arrest, and apoptosis [7]. Heterozygous and homozygous mutations in the RAD50 gene have been reported in two patients with a NBS-like disorder (NBSLD [OMIM 613078]) phenotype, who exhibit microcephaly, growth retardation, and reduced ATM kinase signaling [8,9]; however, it is difficult to evaluate the pathophysiology of RAD50 mutations in humans because these mutations are rare. Therefore, accumulation of patient information on a large scale is required to elucidate the health risks associated with RAD50 mutations in humans. Furthermore, several studies using experimental animals have made it difficult to determine the biological effects of mutations in the rad50 gene. For instance, early mouse embryos that are homozygous for mutated rad50 alleles are lethal [10], which differs from an NBSLD patient with a homozygous mutation of the RAD50 gene [9]. Mice with hypomorphic RAD50 present with hydrocephalus, defects in primitive hematopoietic and gametogenic cells, and liver tumorigenesis [11]. Moreover, this phenotype is not similar to that of NBSLD patients with a mutation in the RAD50 gene [8]. Therefore, further validation is required to elucidate the significance of mutations in the rad50 gene using experimental animals.
Mutations in ATM, MRE11A, and NBS1 genes lead to similar pathogenesis in humans. Comparing these pathogeneses will aid in elucidating the significance of RAD50 mutations. Additionally, these mutations have been recognized as a cause of hereditary and/or malignant diseases in humans. Germline mutations in ATM and MRE11A underlie the autosomal recessive ataxia-telangiectasia syndrome (A-T [OMIM 208900]) and ataxia-telangiectasia-like disorder 1 (ATLD1 [OMIM 604391]), respectively. A-T is characterized by ataxia, telangiectasia, immune defects, chromosomal breakage, predisposition to malignant tumors, and susceptibility to ionizing radiation (IR) [12,13]. Mice carrying homozygous atm mutations exhibit behavioral abnormalities, aberrations in cerebellar Purkinje neurons, and thymic lymphomas, but not telangiectasia [14]. Although patients with ATLD1 also display some similar signs to those with A-T (ataxia and susceptibility to IR), telangiectasia and immune deficiency are not present in these patients [15]. Homozygous mre11a mutations in mice result in embryonic lethality [16]. Germline homozygous mutations in NBS1 in humans are responsible for NBS (OMIM 251260), which is characterized by microcephaly, growth retardation, immunodeficiency, predisposition to cancer, IR-sensitivity, and chromosomal instability in homozygous mutants [17]; in contrast, homozygous mutations in mice result in embryonic lethality [18].
Patients with A-T, ATLD1, and NBS show a predisposition to cancers, including breast cancer, bladder cancer, melanoma, lung cancer, colon cancer, leukemia, and lymphoma, thereby implying the tumorigenic effects of mutations in ATM, MRE11A, and NBS1 genes. We recently identified a heterozygous RAD50 I505fs*5 frameshift germline mutation in a 37-yearold man who presented with simultaneous duodenal and rectal cancers. Genomic analysis of the two cancers in the patient revealed a high tumor mutation burden and high microsatellite instability (details of the clinical and molecular features of the patient will be described elsewhere), suggesting that mutations in the RAD50 gene predispose to cancers, as do mutations in ATM, MRE11A, and NBS1 genes. To demonstrate whether the RAD50 mutation identified in our patient contributed to the development of cancer, we generated and analyzed medaka with mutations in the corresponding exon of the rad50 gene as that in the patient.
Medaka fish (Oryzias latipes) contain genes involved in basic DNA repair pathways in common with vertebrates; thus, they have often been used to detect toxic substances in water [19,20]. These fish have also been used as model organisms to study genes associated with carcinogenic mechanisms [21][22][23] and to elucidate pathways involved in genome homeostasis, such as the p53-mediated damage recognition pathway [24,25] and the base excision repair pathway [26,27]. Compared with mammalian models, fish models are advantageous because of their relatively short lifespans and rapid generation turnover. Previous studies have shown that zebrafish embryogenesis requires DNA repair mechanisms involving the MRN complex and ATM kinases [28] and that medaka fish with nbs1 gene polymorphisms collected from the wild population exhibit defective DSB repair abilities [29]. Therefore, the use of medaka models presents a promising approach to understanding the role of the MRN complex in DSB repair.
In this study, we aimed to 1) precisely understand the clinical significance of RAD50 mutations and determine their role in tumorigenesis; 2) compare our mutant model with existing animal models. We successfully generated multiple tumors and typical A-T phenotypes with corresponding histological abnormalities, telangiectasia, immunological abnormalities, and ataxia in rad50 mutant medaka using the CRISPR/Cas9 system. We also demonstrated that the mutant medaka had a shorter lifespan than the wildtype medaka.

Fish and its husbandry
The medaka STIII strain used to generate medaka models of rad50 deficiency was provided by the National BioResource Project Medaka (https://shigen.nig.ac.jp/medaka/). The STIII medaka fish is transparent because it is recessive to all pigments contained in the chromatophore; thus, it is also termed "transparent medaka." Its transparency allows the internal organs of live fish to be observed [30]. STIII does not show any organ defects other than the loss of pigmentation unless environmentally or genetically manipulated. Fish were reared under a 14 h:10 h light: dark photoperiod at 25-27˚C in a tank with an aquatic filter (Tetra Auto One-Touch Filter AT-20, Spectrum Brands Japan Co. Ltd., Yokohama, Kanagawa, Japan). The water was filtered at a regular speed of 75 L/h, and water condition was maintained at pH 7.2-8.0, NO 3− < 25 mg/L, and dissolved oxygen concentration at 8.50-9.00 mg/L. This study was performed in accordance with the guidelines of the Animal Research Committee of Kyorin University (approval number 235 [for 2019-2021]).

Determination of the nucleotide sequence of STIII medaka rad50
Nucleotide sequence data of human RAD50 (ENSG00000113522) and medaka rad50 (ENSORLG00000001678) were obtained from the Ensembl genome browser (http://www. ensembl.org/). The two sequences were aligned using the CLUSTAL W [31] server at DDBJ (clustalw.ddbj.nig.ac.jp/top-j.html) with default parameters. RAD50 has a coiled-coil structure that can be folded by the hook construct and carries MRE11-and NBS1-binding sites in its structure. The hook domain, a highly conserved homodimerization interface, performs an essential function in the MRN complex by recognizing damaged DNA and promoting DNA repair [1]. Thus, the sequence corresponding to the coiled-coil region before the hook construct of the medaka rad50 was estimated to be the putative exons 5-13. Next, the nucleotide sequences of intron 10, exon 11, intron 11, and exon 12 in rad50 of STIII medaka were determined with an ABI Prism 3100-Avant genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using the primers shown in S1 Table.

Mutant generation
The RAD50 mutation identified in our patient with two cancers was a 2 bp deletion (c.1515_1516 del AA, p.I505fs*5) within exon 8 of the gene on chromosome 5q; exon 8 of human RAD50 corresponds to the putative exon 11 of medaka rad50. The chromosomal location information of the medaka rad50 is not shown in the Ensembl genome browser. Therefore, to generate the rad50 mutant used in this study, guide RNAs; (5 0 -AGUUCAAAGCUCCAAUGUGG-3´) targeting exon 11 of rad50 were designed using CCTop (https://crispr.cos.uni-heidelberg.de/) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). The guide RNA (gRNA) was mixed with tracrRNA (Alt-R CRISPR-Cas9 tracrRNA, IDT) and Cas9 protein (Alt-R S.p. Cas9 Nuclease V3, IDT) and injected into onecell-stage medaka embryos. The injected mixture comprised 3 μM gRNA, 3 μM tracrRNA, and 10 μg/L Cas9 protein solutions (ratio 4.5:4.5:1). The microinjection apparatus used was a Nanoject II (Drummond Scientific Co., Broomall, PA, USA) and 1 mm glass capillaries (Narishige, Tokyo, Japan, G-1) formed using a horizontal pipette puller. Injected embryos were cultured at 28˚C for approximately 10 d, after which the hatched larvae were genotyped to establish stable mutant strains using genomic DNA extracts from their membranous fins, as described in a previous study [32].

Histological analysis
Medaka suspected of being unable to survive till 40-60 weeks of age (3 wildtype and 10 rad50 Δ2/+ and 2 rad50 Δ9/+ medaka from the G 0 and F 1 generations) were immediately euthanized using 0.06% tricaine methane-sulfonate [33] and examined histopathologically. Fish used in the rheotaxis analysis were randomly selected (six wildtype and rad50 Δ2/+ medaka each) and examined. Fish were fixed in Bouin's fluid (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and shaken at room temperature (23-26˚C) for 48 h, following which the solution was changed to a 10% formalin neutral buffer solution and shaken again at room temperature for 24 h. Fixed fish were dehydrated in absolute ethanol and separated into head and trunk parts using a surgical knife. Each part was embedded in paraffin. The eyes, thyroid glands, and brains were observed using coronal sections from the head parts. To observe internal organs, sagittal sections were obtained from the trunks and stained using hematoxylin-eosin. Digital images were captured using AS ONE microscope and AS ONE PCM500 digital camera (AS ONE Co. Ltd., Osaka, Japan).

Survival curve
Wildtype (n = 7, control for G 0 generation; n = 15, control for F 1 generation; n = 17, control for F 2 generation), rad50 Δ2/+ (n = 7 for G0 generation; n = 16 for F 1 generation; n = 33 for F 2 generation), rad50 Δ9/+ (n = 10 for F 1 generation), and rad50 Δ2/Δ2 (n = 18 for F 2 generation) medaka were reared in each of 4.5 L aquaria with a density of up to 10 fish per aquarium. Feed was provided thrice a day, with sufficient feed for all fish to consume in 15 min without any feed remaining at the bottom of the aquarium. Fish that died within 70 weeks from hatching were recorded. Some rad50 Δ2/+ medaka older than 40 weeks showed abnormal swimming with enlargement of the thyroid gland and capillary congestion inside eyes. These fish, which found it difficult to survive and most died within a week, were classified as "dying fish." Samples from fish aged 40-60 weeks were fixed as previously described in histological analysis.

Rheotaxis analysis
Rheotaxis was performed according to Takeuchi et al. [34]. Wildtype (n = 12) and rad50 Δ2/+ medaka from F 3 generation (n = 13) were used for a quantitative comparison of rheotaxis. The intensities of the amplitude of the left and right body movement (body position) and left and right head movement around the center of the body (head yawing) under constant water flow were used as indices. Water flow velocity based on standard body length [SL, the length of a fish measured from the tip of the snout to the posterior end of the last vertebra; no significant difference between SL wildtype (32.7 ± 3.0 mm) and SL mutant (33.3 ± 2.8 mm)), Mann-Whitney U test; p = 0.6444), and 2 × and 3 × SL/s were used for this analysis. Twenty-week-old medaka with no pathological findings in the eyes or internal organs were placed in a swim-mill chamber (10 cm along the y-axis, parallel to the flow direction, and 2 cm along the x-axis), and the flow velocity was set from 2 to 3 × SL/s. Swimming at each velocity was recorded for 10 s using a video recorder (DMK27BUR0135, The Imaging Source, Greenville, SC, USA) located above the chamber (Fig 4A). Positional information of the medaka was used as the output, with the swimming direction as the x-axis and the direction perpendicular to the swimming direction as the y-axis. Next, the change in the y-axis for 10 s was graphed ( Fig 4B) as an example, and the change in the y-axis was calculated as a root mean square (RMS) value. Body and head positions were tracked using DeepLabCut ver. 2.0. [35,36], and their coordinates were extracted (S1 Information). The RMS value of the body position was calculated based on the coordinates of the body positions. For head yawing, the RMS value was calculated based on the coordinates of head positions minus those of body positions.

Measurement of hindbrain size
The size of the hindbrain was quantitatively compared using images of the dorsal side of the brain. After anesthetizing medaka with 0.06% MS-222, the frontal bone was removed using scissors and tweezers to observe the telencephalon, midbrain, and hindbrain. The brains were photographed using a stereomicroscope with a digital camera (Stemi 305, Zeiss, Oberkochen, Germany). Next, the areas of the telencephalon, midbrain, and hindbrain in the image were calculated using ImageJ software (version 1.53e; Bethesda, MD, USA). The area of the hindbrain/(area of the telencephalon + midbrain + hindbrain) was then calculated and used as an index value for a size assessment of each medaka hindbrain.

Measurement of Purkinje cell number
Six wildtype and rad50 Δ2/+ medaka each were randomly selected from the medaka used for rheotaxis analysis to prepare hematoxylin-eosin (HE)-stained specimens of the hindbrain. Purkinje cells in a 400 μm square range, including the boundary between the Purkinje cell layer and the granular layer on the specimens, were counted ( Fig 5A).

Statistical analysis
R software (version 4.0.1) was used for statistical analyses. The chi-square test was used for comparative analysis of the genotype of the offspring from rad50 Δ2/+ crosses and the percentages of Purkinje cells in the granular layers of the hindbrain. Differences in RMS values and hindbrain size between wildtype and rad50 Δ2/+ medaka were analyzed using the Mann-Whitney U test. Changes in the number of surviving fish were estimated using Kaplan-Meier survival curves, whereas the difference in survival curves was analyzed using the log-rank test together with the Bonferroni correction. Differences between the averages of 50% survival times of wildtype and rad50 Δ2/+ medaka were analyzed using Welch's t-test. Statistical significance was set at p < 0.05.

Mutants
The exon composition of the medaka rad50, based on a previous study [1], is illustrated in Fig  1A. With the expectation that the insertion of the mutation downstream of the putative start codon would cause a functional defect, the mutation site was set to the coiled-coil region that precedes the hook construct of medaka rad50. In detail, gRNA corresponding to bases 21-40 of exon 11 of rad50 of STIII medaka was designed (Fig 1B: two-way arrow and double underline). Of the 38 embryos injected with a mixed solution of gRNA and Cas9 nuclease, 24 developed and hatched normally. Mutations in medaka rad50 were detected in 19 of the hatched larvae, and juveniles with 2-or 9-base pair deletion heterozygotes (rad50 Δ2/+ or rad50 Δ9/+ ) were selected and raised. The gene with the 2-base pair deletion was estimated to have a stop codon between the bases 85 and 87 of exon 11 (Fig 1B; asterisk of 'Δ2'), which led to a lack of the hook construct required for rad50 dimer formation causing a functional deficiency. The gene with a 9-base pair deletion (Fig 1B; 'Δ9') was expected to express rad50, which would lack three amino acids but still partly retain its function.

Phenotype of mutants
No difference in growth was observed among the three groups, and no significant difference was observed in the body weights of the dead medaka at 40-60 weeks (326.0 ± 10.5 mg for wildtype [n = 5]; 329.1 ± 81.1 mg for rad50 Δ2/+ medaka [n = 13]; 312.0 ± 38.4 mg for rad50 Δ9/+ medaka [n = 4]; p = 0.9046; one way-ANOVA). Further, 13 out of the 23 rad50 Δ2/+ mutant fish aged 40-60 weeks (n = 7 for the G 0 generation; n = 16 for the F 1 generation) were identified as either dead or dying. All dying rad50 Δ2/+ medaka were either lying on the bottom of the water or swimming abnormally. Some had capillary congestion inside the eyes and enlarged thyroid glands. No obvious abnormalities were observed among the wildtype and rad50 Δ9/+ medaka.

Histological findings
Macro-and microscopic histological findings of rad50 Δ2/+ mutants that had died or were dying at ages 40-60 weeks are shown in Table 1 and described below. Furthermore, one or both eyes of 4 out of the 13 dead or dying fish were swollen (Fig 2A: right eye is shown). A reddish mass was observed at the base of each swollen eye (Fig 2A; arrowhead). Capillary hemangiomas were formed around the choroids of these eyes in 2 out of 10 histologically analyzed fish. Hemangiomas were also observed in the retina (Fig 2C and 2E; arrows #1). Telangiectasias were observed in the hemangiomas and near the pigment epithelial layer (Fig 2C and 2E; arrows #2). The structures of these retinas collapsed (Fig 2C), and many histiocytes were scattered between the rod/pyramidal cell layer and the pigment epithelial layer (Fig 2C and 2E; arrow #3). The thyroid glands of 4 out of the 10 histologically analyzed fish of rad50 Δ2/+ mutants are shown in Fig 2B (arrowhead). Four fish showed proliferation by follicle epithelial cells (Fig 2F; arrow), which formed nodular thyroid hyperplasia (Fig 2H). In addition, some granulomatous nodules (Fig 2I and 2J; arrow #1) and many histiocytes (Fig 2J; arrow #2) were observed in the livers of three fish. Granulomas (Fig 2L and 2M; arrows #1) and hemangiomas (Fig 2E, arrow #2; Fig 2N, magnified view) were observed in the kidney, and telangiectasias were observed in the hemangiomas. A structure dominated by adrenal gland-derived cells was observed in the kidneys of one of the 10 histologically analyzed fish (Fig 2O). Proliferated adrenal-derived cells (Fig 2P; adrenal cortex-derived cells indicated by arrow #1 and adrenal medulla cells indicated by arrow #2) and infiltrated histiocytes (Fig 2P; arrow #3) were observed in this structure. Thus, one or two of the above-mentioned tumorous lesions were observed in 8 out of the 10 histologically analyzed fish of the rad50 Δ2/+ mutants and telangiectasias in 6 of the mutant fish. Three of the wildtypes and two of the rad50 Δ9/+ mutants were observed histologically, but no histological abnormalities were observed (Fig 2D, 2G, 2K and 2Q).

Survival time
Survival curves corresponding to a period of 70 weeks after hatching of wildtype, rad50 Δ2/+ , and rad50 Δ9/+ medaka in the F 1 generation were compared. A significant difference was observed among the three curves (Fig 3A; n = 15 for wildtype; n = 16 for rad50 Δ2/+ ; n = 10 for rad50 Δ9/+ ; p = 0.002, log-rank test with Bonferroni correction), as well as between the median survival times for wildtype, rad50 Δ9/+ , and rad50 Δ2/+ medaka (66.5, 65.5, and 56.0 weeks, respectively). The survival curve of rad50 Δ9/+ medaka was similar to that of wildtype. Additionally, in the other generations (G 0 and F 2 ), the number of dead or dying rad50 Δ2/+ fish increased at around 40 weeks of age, and significant differences were observed between the survival times of wildtype and rad50 Δ2/+ groups (p < 0.01; log-rank test). The average median survival times of G 0 , F 1 , and F 2 generations of both wildtype and rad50 Δ2/+ medaka were 65.7 ± 1.1 and  The ratio of the number of larvae from in-crosses of rad50 Δ2/+ medaka was wildtype:rad50 Δ2/+ :rad50 Δ2/Δ2 = 22:46:18, respectively, which was not significantly different from the Mendelian ratio (p = 0.734; Chi-square test). However, the homozygous mutants hardly survived after 4 weeks of hatching (Fig 3B), and their 50% survival time was 6 weeks. The swimming was unstable even in rearing water without water flow. The reduced lifespan of rad50 Δ2/Δ2 homozygotes made it difficult to maintain the samples before analyses. rad50 Δ9/+ medaka were subjected to survival time measurement and histological analyses to determine whether genome-editing frameshift mutations without putative premature stop codons affected lifespans and tumorigenesis. However, these homozygotes could not be analyzed in detail because of their smaller sample sizes. In-crosses of rad50 Δ9/+ medaka produced only three rad50 Δ9/Δ9 medaka with 63, 68, and 70 weeks or more lifespans, which were close to those of wildtype medaka, suggesting that the 9-base pair deletion did not affect the lifespan of medaka.

Rheotaxis analysis
Rheotaxis abilities were quantitatively compared to investigate the effect of the 2-base pair deletion of rad50 on medaka hindbrains. This was based on the fact that fish with normal hindbrains are able to maintain their body position while swimming under constant water flow conditions. At a flow velocity of 2 × SL/s, the RMS value of rad50 Δ2/+ medaka was significantly higher than that of wildtype medaka (Fig 4C; Mann-Whitney U test; p = 0.036). However, with increased flow velocity (3 × SL/s), the difference between the RMS values of rad50 Δ2/ + and wildtype medaka became greater (Mann-Whitney U test; p < 0.001), indicating that rad50 Δ2/+ medaka were inferior to wildtype medaka in terms of rheotaxis ability. With respect to head yawing at a flow velocity of 2 × SL/s, no significant difference was observed between the RMS values of rad50 Δ2/+ and wildtype medaka (Fig 4D; Mann-Whitney U test; p = 0.8065). However, at a flow velocity of 3 × SL/s, the RMS value of rad50 Δ2/+ medaka was significantly higher than that of wildtype medaka (Mann-Whitney U test; p < 0.001). S1 and S2 Movies show representative wildtype and rad50 Δ2/+ medaka, respectively, swimming at a velocity of 3 × SL/s. There was no significant difference between rad50 Δ2/+ and wildtype medaka at a flow velocity of 1 × SL/s. The medaka could not swim for 10 s under a flow velocity of 4 × SL/s.

Discussion
To the best of our knowledge, this is the first study to successfully generate a model fish carrying mutant rad50. To ensure a null mutation of medaka rad50, we used genome editing to target the position of the coiled-coil region preceding the hook construct to generate a stop codon close to the back of that position. With the loss of the hook construct, rad50 stopped expressing functional RAD50 [11,[37][38][39]. Therefore, the 2-base pair deletion in rad50 of rad50 Δ2/+ medaka would have resulted in the loss of functional rad50. In contrast, a 9-base pair deletion in rad50 that caused a small in-frame deletion of rad50 retained some rad50 function in rad50 Δ9/+ medaka. Furthermore, STIII medaka is a transparent fish in which morphological abnormalities, including organ defects and tumorigenesis, can be easily observed. Therefore, rad50 Δ2/+ and rad50 Δ9/+ STIII medaka were deemed to be appropriate models that could be used to evaluate the phenotypes affected by rad50 mutations. All rad50 Δ2/Δ2 medaka were unstable in swimming even under normal conditions without water flow, suggesting the possibility that the rheotaxis ability of the homozygous mutants was lower than that of heterozygotes. On the contrary, no abnormalities in swimming and lifespan were observed in rad50 Δ9/ Δ9 medaka in a normal rearing condition, although those with a small sample size were preliminarily analyzed, suggesting that the 2 bp deletion is more harmful than the 9 bp deletion in terms of swimming abilities and lifespan of medaka. Given the short survival time and small sample sizes of the homozygotes, the effects of rad50 frameshift mutation with premature translation stop on tumorigenesis, lifespan, A-T, etc., were mainly discussed based on the comparison between wildtype and rad50 Δ2/+ medaka. Capillary hemangiomas of the retina, kidney, and nodular thyroid hyperplasia were observed in rad50 Δ2/+ medaka. These results may substantiate the reported tumorigenic properties of RAD50, as suggested by the prevalence of RAD50 mutations in patients with cancer. A study on Chinese patients with breast cancer demonstrated no association between RAD50 mutation and the morbidity associated with the disease [40]. In contrast, a study in Finnish patients with breast and ovarian cancer reported a significant association between RAD50 687delT and predisposition to cancer in Finnish people [41]. Concordant with this previous study [41], we identified a RAD50 I505fs germline nonsense mutation in the RAD50 gene, indicating an association of RAD50 mutation with the cancers in the 37-year-old patient presented with concomitant duodenal and rectal cancers. The exon containing the mutation in the patient corresponds to the position where the stop codon was induced using the CRISPR/ Cas9 system in the current study. Therefore, our medaka mutant demonstrated that, as shown by experimental animals with other defective MRN-ATM pathway molecules, the loss-of-function mutation of rad50 would have tumorigenic properties; however, further studies are required to elucidate the clinical significance of the RAD50 mutation in human cancers.
Epidemiological studies have indicated that mutations in DNA repair genes are involved in lifespan and aging [42]. However, evidence regarding the effect of RAD50 mutations on lifespan is inadequate. Mouse early embryonic stem cells homozygous for mutated alleles of rad50 are nonviable [10]; the study showed no distinctive features regarding growth rate, viability, and fertility in mice heterozygous for the mutant allele compared to wildtype. Bender et al. (2002) demonstrated a decrease in growth rate but did not show the lifespan data of the mouse hypomorphic RAD50 model [43]. The results of the current study indicated that the survival time of heterozygous mutants was shorter than that of wildtype medaka, even in environments free of exposure to mutagens, such as UV light and carcinogens. The short lifespan could be attributed to the rad50 mutation and/or mutation-associated histological changes occurring after approximately 40 weeks of age after hatching. The hatched larvae from a cross between heterozygotes exhibited a Mendelian genotypic ratio. As opposed to zebrafish studies that demonstrated that embryonic development was affected by rad50 [28], our results suggest that homozygous mutations in rad50 may not affect embryogenesis and hatching in a stable environment. However, normal rad50 appears essential for survival in the early stages of development following hatching because the survival time in homozygous mutants was considerably shorter after hatching than before.
RAD50 mutations were detected in two patients with partial symptoms of NBS, classified as NBSLD [8,9]. Mutations in RAD50 will arguably lead to some of the same symptoms of A-T, as do ATLD1 and NBS, having been caused by mutations in other components of the MRN/ ATM pathway. Histological analysis in rad50 Δ2/+ medaka showed an association between rad50 and telangiectasia. Telangiectasia, which are found on the surface of the skin or the edges of eyeballs [13], have been observed in patients with A-T [12]; however, telangiectasia have not been previously observed in experimental animals with mutations in atm, mre11a, or nbs1 genes [11,43]. To the best of our knowledge, rad50 Δ2/+ medaka is the first experimental animal model exhibiting telangiectasia, similar to that found in patients with A-T. In this study, the rheotaxis ability against flow velocity was used as an evaluation index for ataxia. The hindbrain of fish, which corresponds to the cerebellum of mammals, integrates processes such as vestibular motor reflexes, gaze retention, and lateral line signal detection to maintain posture against flow velocity [44][45][46]. Several experiments using zebrafish and medaka have demonstrated that deficiencies in the genes required for hindbrain homeostasis may reduce rheotaxis ability [34]. Mice with disordered Purkinje and granular cells in the cerebellum showed poor performance in the rotarod test, which measures coordination and motor learning [47]. Therefore, disordered cells in the rad50 Δ2/+ medaka hindbrain may lead to deterioration of rheotaxis ability. In addition, spindle-shaped Purkinje cells that were not observed in the wildtype were characteristically observed in rad50 Δ2/+ medaka. The proximal axons of Purkinje cells in the cerebellum swell into spindle-shaped Purkinje cells (torpedo) in patients with A-T [48], suggesting that the torpedo in rad50 Δ2/+ medaka may also be associated with defects in their rheotaxis ability. These results demonstrated that in vertebrates, associations exist between the rad50 mutation and ataxia or telangiectasia.
Histological analysis in our mutant supported data associated with immunosuppression in mice with hypomorphic RAD50 [11]. Although no immune impairment has been observed in the two patients with NBSLD, some patients with A-T show immune disorders, and mutant mice with hypomorphic RAD50 showed defects in primitive hematopoietic cells [11,43].
Histiocyte and lymphocyte infiltration in rad50 Δ2/+ medaka was the abnormal proliferation of cells involved in the lymphatic system. A-T is strongly associated with the abnormal proliferation of cells involved in the lymphatic system, as proliferation weakens the immune system [49]. Our study demonstrated that rad50 Δ2/+ medaka showed immunosuppression, which constitutes a part of the findings in patients with A-T. No histological changes were observed in reproductive tissues, and no abnormal mating behavior was observed in rad50 Δ2/+ medaka, and the embryogenesis of offspring was normal.
Our study has a few limitations. First, the size of the analyzed cohorts was relatively small. Therefore, further studies with larger sample sizes are warranted. Second, although A-T, ATLD1, NBS, and NBSLD are typically autosomal recessive disorders associated with biallelic mutations of a relevant gene, most results of the present study are based on an analysis of heterozygotes of rad50 mutation. However, a subset of A-T patients carries a heterozygous ATM mutation, suggesting that some type of heterozygous ATM deficiency may give rise to the A-T phenotype. Similarly, RAD50 mutants may exert haploinsufficiency or dominant-negative effects on A-T phenotype development. A previous study also implicated the dose-dependent dominance of a heterozygous rad50 mutant in a mouse model [10]. In our medaka model, the homozygous mutant of rad50 was semi-lethal and, thus, inappropriate for the precise analysis of several data sets. Regardless of the zygosity of the mutation, our medaka model exhibiting the A-T phenotype will be useful in future experiments investigating the pathogenesis of the disease.
In conclusion, the analysis of medaka fish revealed that a heterozygous mutation of rad50 could cause tumorigenesis. Furthermore, the mutation results in a short lifespan, which is one of the signs of A-T, ATLD1, NBS, and NBSLD patients. In addition, to the best of our knowledge, this study is the first to demonstrate that most A-T phenotypes may be concurrently reproduced via a vertebrate rad50 germline mutation model. The rad50 Δ2/+ STIII medaka developed ataxia represented by rheotaxis disability, Purkinje cell disorder in the hindbrain, telangiectasia, immunological abnormalities, and tumor formation, all of which are typical characteristics of A-T patients. Thus, rad50 Δ2/+ medaka could contribute to a deeper understanding of the molecular mechanisms underlying RAD50 mutation-induced tumors and related A-T phenotype, as well as help develop novel therapeutic strategies based on improved insight into molecular disorders.
Supporting information S1 Fig. Hindbrain size. A ratio of the area of the hindbrain to area of the telencephalon, midbrain, and hindbrain in wildtype (WT) and rad50 Δ2/+ medaka. The areas were calculated using images. The black dots and short horizontal red lines represent the data obtained from one individual and the median, respectively. n.s. indicates the absence of a statistically significant difference (Mann-Whitney U test). (TIF) S1 Table. PCR and sequencing primers. (DOCX) S1 Information. Creation of an analytical model and extraction of the head and body coordinates of swimming medaka from movies via DeepLabCut. DeepLabCut (version 2.0) was used to create an analytical model of medaka swimming and to extract the coordinates of the head and body positions. First, 19 frames were automatically extracted using the "kmeans" algorithm from the video of one wildtype medaka. In these frames, the head position was labeled between the left and right eyes. The body position was labeled between the left and right pectoral fins. The size of each marker was 5 μm. Nineteen frames were used as the training dataset in DeepLabCut. Next, DeepLabCut was trained 50,000 times to recognize the position of the head and body of the medaka in the movie based on the training dataset, and an analysis model of medaka swimming was generated. The analytical model of medaka swimming was used to analyze all movies of wildtype and rad50 Δ2/+ medaka, and the head and body coordinates were extracted. The root mean square values were calculated from the coordinates using Microsoft Excel. The tail markers shown in the movies were not used in this study.